SLC16A3-Induced Lactate Remodeling Drives Immune Evasion in Clear Cell Renal Cell Carcinoma via an Autocrine GPR81-ERK-c-MYC Feedback Loop.

[BACKGROUND] Extensive studies have implicated glycolytic reprogramming and lactate accumulation in immune evasion. However, clustered regularly interspaced short palindromic repeats (CRISPR)-based screens systematically interrogating metabolic drivers of immune checkpoint blockade resistance in clear cell renal cell carcinoma (ccRCC) remain scarce. Consequently, direct evidence delineating how the lactate exporter solute carrier family 16 member 3 (; monocarboxylate transporter 4, MCT4) rewires tumor lactate metabolism to promote immune escape in ccRCC is still lacking.

[AIMS] To identify key metabolic genes governing immune checkpoint blockade resistance in ccRCC via CRISPR screening, elucidate the molecular mechanisms of -mediated lactate reprogramming in regulating glycolysis and immune suppression, and validate the therapeutic potential of MCT4 inhibition combined with anti-programmed cell death protein-1 (anti-PD-1) therapy for ccRCC.

[STUDY DESIGN] Integrated in vivo CRISPR screening, functional assays, and clinical validation.

[METHODS] A metabolic CRISPR library-based screen was conducted in an immunocompetent ccRCC model treated with an anti-PD-1 antibody. Single-guide RNAs (sgRNAs) targeting glycolysis-associated genes were differentially enriched. Analyses of The Cancer Genome Atlas dataset and the Tumor Immune Estimation Resource database were performed, and an institutional tissue microarray analysis assessed survival outcomes. -knockout and -overexpressing RENCA cells were generated to evaluate tumor growth in immunodeficient and immunocompetent mice, alongside immune cell infiltration profiling. Seahorse metabolic assays, flow cytometry, lactate-treated macrophage assays, and G-protein-coupled receptor 81 (GPR81) antagonism or genetic suppression were used to dissect the lactate export/receptor axis. Additionally, -knockdown and -overexpressing 786-O and SN12C ccRCC cells were established. RNA sequencing, protein stability, ubiquitination, and phosphorylation analyses were conducted to elucidate the underlying molecular mechanisms.

[RESULTS] Anti-PD-1 therapy selectively reduced sgRNA counts for glycolysis-lactate genes, most notably Ldha and . While depletion modestly restricted intrinsic ccRCC proliferation in immunodeficient models, its predominant effect was remodeling the tumor immune microenvironment. -mediated lactate export activated macrophage extracellular signal-regulated kinase (ERK) signaling in a GPR81-dependent manner, promoted M2 macrophage polarization, and suppressed CD8 T-cell function. Concurrently, exported lactate engaged tumor GPR81 to activate autocrine ERK signaling and phosphorylate c-MYC at Ser62, preventing F-box and WD repeat domain-containing 7-mediated degradation. This stabilized c-MYC upregulated lactate dehydrogenase A, glucose transporter 1, and hypoxia-inducible factor 1 alpha, forming a self-sustaining glycolytic feedback loop uniquely amplified in the von Hippel-Lindau-deficient background of ccRCC. Clinically, tissue microarray analysis indicated a trend toward worse progression-free survival in patients with high MCT4 expression. Furthermore, combining the MCT4 inhibitor MSC-4381 with PD-1 blockade markedly reduced tumor volume in immunocompetent mice, demonstrating enhanced combinatorial efficacy.

[CONCLUSION] -induced lactate reprogramming drives immune resistance in ccRCC via two converging mechanisms: extracellular lactate-mediated immunosuppression through M2 macrophage polarization and an autocrine GPR81-ERK-c-MYC glycolytic feedback loop. These findings highlight MCT4 blockade combined with immune checkpoint inhibition as a rational strategy to overcome immunotherapy resistance.